Response Element Regions

ABSTRACT

Response element regions, DNA constructs comprising response element regions, host cells comprising response element regions, and methods of using response element regions are provided.

This application is a continuation of and claims priority to U.S. Ser.No. 11/112,973, filed Apr. 22, 2005, currently pending, which claims thebenefit of U.S. Provisional Application No. 60/564,724, filed Apr. 22,2004; and U.S. Provisional Application No. 60/565,135, filed Apr. 23,2004, all of which are incorporated by reference herein for any purpose.

FIELD

Response element regions, DNA constructs comprising response elementregions, host cells comprising response element regions, and methods ofusing response element regions are provided.

BACKGROUND

In certain cellular systems, certain molecules, such as cytokines andgrowth factors, can interact with a receptor of a cell, which triggers aprocess that affects the activity of one or more transcription factor.In certain instances, the process activates one or more transcriptionfactors, which then bind to a response element region of a gene toinduce transcription. In certain instances, the transcription factor isa Signal Transducer and Activator of Transcription (STAT).

SUMMARY OF THE INVENTION

In certain embodiments, an isolated nucleic acid comprising a responseelement region is provided. In certain embodiments, a vector comprisinga promoter and nucleic acid comprising a response element region isprovided. In certain embodiments, a host cell comprising a vector isprovided.

In certain embodiments, a response element region comprises (i) thesequence GTCATTTCCAGGAAATCACC or (ii) a sequence complementary to thesequence in (i).

In certain embodiments, a response element region comprises (i) thesequence GTCATTTCCAGGAAATCACCGTCATTTCCAG GAAATCACCGTCATTTCCAGGAAATCACCor (ii) a sequence complementary to the sequence in (i). In certainembodiments, a response element region comprises (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC or (ii) a sequence complementaryto the sequence in (i). In certain embodiments, a response elementregion comprises (i) the sequence GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC or (ii) a sequence complementary to the sequence in (i).In certain embodiments, a response element region comprises (i) thesequence GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACC or (ii) a sequencecomplementary to the sequence in (i). In certain embodiments, Y, X, andZ are each independently selected from a nucleic acid sequence of 0 to23 nucleotides.

In certain embodiments, a method for determining the activity of a testcomposition comprising a signaling molecule is provided. In certainembodiments, the method comprises contacting the test composition with asignaling molecule-responsive host cell comprising a vector thatcomprises a promoter and nucleic acid comprising a response elementregion. In certain embodiments, the method further comprises incubatingthe test composition comprising a signaling molecule and thesignaling-responsive host cell under conditions in which the reporternucleic acid expresses a reporter protein in response to the signalingmolecule. In certain embodiments, the method further comprises detectingthe reporter protein to determine the activity of the test composition.

In certain embodiments, a method for determining whether a test compoundhas activity of a given signaling molecule is provided. In certainembodiments, the method comprises contacting the test compound with asignaling molecule-responsive host cell comprising a vector thatcomprises a promoter and nucleic acid comprising a response elementregion. In certain embodiments, the method further comprises incubatingthe test compound and the signaling-responsive host cell underconditions in which the reporter nucleic acid expresses a reporterprotein in response to the signaling molecule. In certain embodiments,the method further comprises detecting the reporter protein to determinethe activity of the test composition. In certain embodiments, the methodfurther comprises comparing the level of detected reporter proteinexpression with the level of detected reporter protein expressed by asignaling molecule-responsive host cell comprising the vector in theabsence of the test compound to determine whether the test compound hasthe activity of the given signaling molecule. In certain embodiments,the method further comprises comparing the level of detected reporterprotein expression with the level of detected reporter protein expressedby a signaling molecule-responsive host cell comprising the vector inthe presence of the given signaling molecule, but in the absence of thetest compound to determine whether the test compound has the activity ofthe given signaling molecule.

In certain embodiments, a method for determining whether a test compoundimpacts the activity of a signaling molecule is provided. In certainembodiments, the method comprises contacting the test compound with asignaling molecule-responsive host cell comprising a vector thatcomprises a promoter and nucleic acid comprising a response elementregion in the presence of the signaling molecule under conditions inwhich the reporter nucleic acid expresses a reporter protein in responseto the signaling molecule. In certain embodiments, the method furthercomprises detecting the reporter protein. In certain embodiments, themethod further comprises comparing the level of detected reporterprotein expression with the level of detected reporter protein expressedby a signaling molecule-responsive host cell comprising the vector inthe presence of the signaling molecule, but in the absence of the testcompound, to determine whether the test compound impacts the activity ofthe signaling molecule.

In certain embodiments, a method of producing a polypeptide from an exvivo mammalian system is provided. In certain embodiments, the methodcomprises producing the polypeptide. In certain embodiments, the methodfurther comprises testing the polypeptide with a signalingmolecule-responsive host cell comprising a vector that comprises apromoter and nucleic acid comprising a response element region. Incertain embodiments, the method further comprises determining the amountof protein produced and/or activity of the protein produced by the exvivo system.

In certain embodiments, a response element region comprising more thanone response element sequences is provided. In certain embodiments, aresponse element sequence comprises the sequence GTCATTTCCAGGAAATCACC.In certain embodiments, the center region of at least two responseelement sequences are spatially oriented to be in the same location (onthe y and z axis) plus or minus 36 degrees, relative to the center axisof the double-helical DNA (x-axis). In certain embodiments, the centerregion is the tenth and eleventh nucleotides AG of the sequenceGTCATTTCCAGGAAATCACC.

In certain embodiments, a response element region comprising more thanone response element sequence core regions is provided. In certainembodiments, a response element sequence core region comprises thesequence TTCCAGGAA. In certain embodiments, the center region of atleast two response element sequence core regions are spatially orientedto be in the same location (on the y and z axis) plus or minus 36degrees, relative to the center axis of the double-helical DNA (x-axis).In certain embodiments, the center region is the fifth and sixthnucleotides AG of the sequence TTCCAGGAA. In certain embodiments, aresponse element region comprising at least two series of more than oneresponse element sequences is provided. In certain embodiments, aresponse element sequence comprises the sequence GTCATTTCCAGGAAATCACC.In certain embodiments, each series of more than one response elementsequences are linked together by a sequence of approximately eightnucleotides. In certain embodiments, within a first series of theresponse element sequences, each center region of the response elementsequences are spatially oriented to be in approximately the samelocation (on the y and z axis) plus or minus 36 degrees, relative to thecenter axis of the double-helical DNA (x-axis). In certain embodiments,the center region is the tenth and eleventh nucleotides AG of thesequence GTCATTTCCAGGAAATCACC. In certain embodiments, within a secondseries of the response element sequences, each center region of theresponse element sequences are spatially oriented to be in approximatelythe same location (on the y and z axis) plus or minus 36 degrees,relative to the center axis of the double-helical DNA (x-axis). Incertain embodiments, the center region is the tenth and eleventhnucleotides AG of the sequence GTCATTTCCAGGAAATCACC. In certainembodiments, the center region of the response element sequences of thesecond series of the response element sequences are spatially orientedto be approximately 72 to 86 degrees from the center region of the firstseries of the response element sequences as determined from the y and zaxis relative to the center axis of the double-helical DNA as the xaxis.

In certain embodiments, a response element region comprising at leasttwo series of more than one response element sequences is provided. Incertain embodiments, a response element sequence comprises the sequenceGTCATTTCCAGGAAATCACC. In certain embodiments, each series of more thanone response element sequences are linked together by a sequence ofapproximately eight nucleotides. In certain embodiments, within a firstseries of the response element sequences, each center region of theresponse element sequences are spatially oriented to be in approximatelythe same location (on the y and z axis) plus or minus 36 degrees,relative to the center axis of the double-helical DNA (x-axis). Incertain embodiments, the center region is the tenth and eleventhnucleotides AG of the sequence GTCATTTCCAGGAAATCACC. In certainembodiments, within a second series of the response element sequences,each center region of the response element sequences are spatiallyoriented to be in approximately the same location (on the y and z axis)plus or minus 36 degrees, relative to the center axis of thedouble-helical DNA (x-axis). In certain embodiments, the center regionis the tenth and eleventh nucleotides AG of the sequenceGTCATTTCCAGGAAATCACC. In certain embodiments, the center region of theresponse element sequences of the second series of the response elementsequences are spatially oriented to be approximately 144 to 180 degrees(in certain embodiments, from 144 to 172 degrees) from the center regionof the first series of the response element sequences as determined fromthe y and z axis relative to the center axis of the double-helical DNAas the x axis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an exemplary representation of the sequenceGTCATTTCCAGGAAATCACC on a double helix DNA. FIG. 1 also depicts the x,y, and z axis.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

The section headings used herein are for organizational purposes only,and are not to be construed as limiting the subject matter described.All documents cited in this application, including, but not limited topatents, patent applications, articles, books, and treatises, areexpressly incorporated by reference in their entirety for any purpose.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transfection (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)). Unless specific definitions are provided,the nomenclatures utilized in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquesmay be used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

A response element region refers to a region of a double-strandednucleic acid that is capable of being bound by one or more activatedresponse element transcription factors, or one or more complexes ofactivated response element transcription factors, to modulate expressionof one or more genes. A response element region also refers to a regionof a single-stranded nucleic acid that, if it were double-stranded DNA,would be capable of being bound by one or more activated responseelement transcription factors, or one or more complexes of activatedresponse element transcription factors, to modulate expression of one ormore genes.

The term response element transcription factors refers to factors thatbind to a response element region to modulate expression of one or moregenes. In certain embodiments, multiple response element transcriptionfactors may bind to a response element region. In certain embodiments,one or more complexes of response element transcription factors may bindto a response element region.

The term “operably linked” refers to components that are in arelationship permitting them to function in their intended manner. Forexample, a control sequence “operably linked” to a coding sequencepermits expression of the coding sequence under conditions compatiblewith the operation of the control sequence.

The term “control sequence” refers to a nucleic acid sequence which mayeffect the expression and processing of coding sequences. According tocertain embodiments, control sequences may include response elementregions and promoters.

A signaling molecule refers to an extracellular molecule, in either afree or bound form, that interacts with a of a cell, which triggers aprocess that affects the activity of one or more response elementtranscription factor.

A signaling molecule-responsive host cell refers to a host cell thatcomprises a signaling molecule receptor capable of interacting with asignaling molecule.

The term “reporter nucleic acid” refers to a nucleic acid that encodes apolypeptide that can be used to detect expression of the reporternucleic acid.

The term “isolated nucleic acid” or “isolated polynucleotide” as usedherein shall mean a nucleic acid of genomic, cDNA, or synthetic originor some combination thereof, which by virtue of its origin the “isolatednucleic acid” (1) is not associated with all or a portion of apolynucleotide in which the “isolated nucleic acid” is found in nature,(2) is linked to a nucleic acid which it is not linked to in nature, or(3) does not occur in nature as part of a larger sequence.

The term “nucleic acid” or “polynucleotide” as referred to herein meansa polymeric form of nucleotides. In certain embodiments, the bases maycomprise at least one of ribonucleotides, deoxyribonucleotides, and amodified form of either type of nucleotide. The term includes single anddouble stranded forms of DNA.

The term “naturally occurring nucleotides” includes deoxyribonucleotidesand ribonucleotides. Deoxyribonucleotides include, but are

not limited to, adenosine, guanine, cytosine, and thymidine.Ribonucleotides include, but are not limited to, adenosine, cytosine,thymidine, and uricil. The term “modified nucleotides” includesnucleotides with modified or substituted sugar groups and the like. Theterm “oligonucleotide linkages” includes oligonucleotides linkages suchas phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See, e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984);Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). In certain instances,an oligonucleotide can include a label for detection.

The term “polypeptide” is used herein as a generic term to refer to anypolypeptide comprising two or more amino acids joined to each other bypeptide bonds or modified peptide bonds, i.e., peptide isosteres.“Polypeptide” refers to both short chains, commonly referred to aspeptides, oligopeptides or oligomers, and to longer chains, generallyreferred to as proteins. Polypeptides may contain amino acids other thanthose normally encoded by a codon.

Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.Such modifications may be present to the same or varying degrees atseveral sites in a given polypeptide. Also, in certain embodiments, agiven polypeptide may contain many types of modifications such asdeletions, additions, and/or substitutions of one or more amino acids ofa native sequence. In certain embodiments, polypeptides may be branchedas a result of ubiquitination, and, in certain embodiments, they may becyclic, with or without branching. Cyclic, branched and branched cyclicpolypeptides may result from post-translation natural processes or maybe made by synthetic methods. Modifications include, but are not limitedto, acetylation, acylation, ADP-ribosylation, amidation, biotinylation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

Identity and similarity of related polypeptides can be readilycalculated by known methods. Such methods include, but are not limitedto, those described in Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York (1988); Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York (1993);Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press (1987); SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press,New York (1991); and Carillo et al., SIAM J. Applied Math., 48:1073(1988). A protein is “substantially similar” to another protein, as itis meant herein, when it is at least 90% identical to the other proteinin amino acid sequence and maintains or alters in a desirable manner thebiological activity of the unaltered polypeptide. Biological activitycan be measured by an in vivo assay such as that described by Cotes andBangham ((1961), Nature 191: 1065-67).

Certain methods to determine identity are designed to give the largestmatch between the sequences tested. Certain, methods to determineidentity are described in publicly available computer programs. Certaincomputer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package, including GAP(Devereux et al., Nucl. Acid. Res., 12:387 (1984); Genetics ComputerGroup, University of Wisconsin, Madison, Wis., BLASTP, BLASTN, and FASTA(Altschul et al., J. Mol. Biol., 215:403-410 (1990)). The BLASTX programis publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra (1990)). Thewell-known Smith Waterman algorithm may also be used to determineidentity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in certain embodiments, the selected alignmentmethod (GAP program) will result in an alignment that spans at least 50contiguous amino acids of the target polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). In certain embodiments, a gap openingpenalty (which is calculated as 3× the average diagonal; the “averagediagonal” is the average of the diagonal of the comparison matrix beingused; the “diagonal” is the score or number assigned to each perfectamino acid match by the particular comparison matrix) and a gapextension penalty (which is usually 1/10 times the gap opening penalty),as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used inconjunction with the algorithm. In certain embodiments, a standardcomparison matrix (see Dayhoff et al., Atlas of Protein Sequence andStructure, 5(3)(1978) for the PAM 250 comparison matrix; Henikoff etal., Proc. Natl. Acad. Sci USA, 89:10915-10919 (1992) for the BLOSUM 62comparison matrix) is also used by the algorithm.

In certain embodiments, the parameters for a polypeptide sequencecomparison include the following:

Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970);

Comparison matrix: BLOSUM 62 from Henikoff et al., supra (1992);

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program may be useful with the above parameters.

In certain embodiments, the aforementioned parameters are the defaultparameters for polypeptide comparisons (along with no penalty for endgaps) using the GAP algorithm.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include, but are not limited to: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded nucleic acid sequences is the 5′ end; the left-handdirection of double-stranded nucleic acid sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences.”

Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Naturally occurring residues may be divided into classes based on commonside chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In certain embodiments, those which arewithin ±1 are included, and in certain embodiments, those within ±0.5are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 1.

TABLE 1 Amino Acid Substitutions More specific Original Exemplaryexemplary Residues Substitutions Substitutions Ala Val, Leu, Ile Val ArgLys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn AsnGlu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val,Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala,Phe Lys Arg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, IleLeu Phe Leu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys ThrThr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met,Leu, Phe, Leu Ala, Norleucine

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth herein using well-known techniques. In certainembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In certainembodiments, one can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In certain embodiments,even areas that may be important for biological activity, including butnot limited to the CDRs of an antibody, or that may be important forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a polypeptide that correspondto amino acid residues which are important for activity or structure insimilar polypeptides. One skilled in the art may opt for chemicallysimilar amino acid substitutions for such predicted important amino acidresidues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of a polypeptide withrespect to its three dimensional structure. In certain embodiments, oneskilled in the art may choose not to make radical changes to amino acidresidues predicted to be on the surface of the polypeptide, since suchresidues may be involved in important interactions with other molecules.Moreover, one skilled in the art may generate test variants containing asingle amino acid substitution at each desired amino acid residue. Thevariants can then be screened using activity assays known to thoseskilled in the art. Such variants could be used to gather informationabout suitable variants. For example, if one discovered that a change toa particular amino acid residue resulted in destroyed, undesirablyreduced, or unsuitable activity, variants with such a change may beavoided. In other words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech.,7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974);Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann.Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural database (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will become dramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al.,Structure, 4(1):15-19 (1996)), “profile analysis” (Bowie et al.,Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159(1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358(1987)), and “evolutionary linkage” (See Holm, supra (1999), andBrenner, supra (1997)).

In certain embodiments, antibody variants include glycosylation variantswherein the number and/or type of glycosylation site has been alteredcompared to the amino acid sequences of the parent polypeptide. Incertain embodiments, polypeptide variants comprise a greater or a lessernumber of N-linked glycosylation sites than the native polypeptide. AnN-linked glycosylation site is characterized by the sequence: Asn-X-Seror Asn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionswhich eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created.

In certain embodiments, polypeptide variants include cysteine variants.In certain embodiments, cysteine variants have one or more cysteineresidues that are deleted from or that are replaced by another aminoacid (e.g., serine) as compared to the parent amino acid sequence. Incertain embodiments, cysteine variants have one or more cysteineresidues that are added to or that replace another amino acid (e.g.,serine) as compared to the parent amino acid sequence. In certainembodiments, cysteine variants may be useful when polypeptides arerefolded into a biologically active conformation such as after theisolation of insoluble inclusion bodies. In certain embodiments,cysteine variants have fewer cysteine residues than the nativepolypeptide. In certain embodiments, cysteine variants have morecysteine residues than the native polypeptide. In certain embodiments,cysteine variants have an even number of cysteine residues to minimizeinteractions resulting from unpaired cysteines.

According to certain embodiments, amino acid substitutions are thosewhich: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter binding affinities, and/or (4) confer ormodify other physicochemical or functional properties on suchpolypeptides. According to certain embodiments, single or multiple aminoacid substitutions (in certain embodiments, conservative amino acidsubstitutions) may be made in the naturally-occurring sequence (incertain embodiments, in the portion of the polypeptide outside thedomain(s) forming intermolecular contacts). In certain embodiments, aconservative amino acid substitution typically may not substantiallychange the structural characteristics of the parent sequence (e.g., areplacement amino acid should not tend to break a helix that occurs inthe parent sequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion. In certainembodiments, fragments are at least 5 to 467 amino acids long. It willbe appreciated that in certain embodiments, fragments are at least 5, 6,8, 10, 14, 20, 50, 70, 100, 150, 200, 250, 300, 350, 400, or 450 aminoacids long.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987). Such compounds are often developed with theaid of computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce a similar therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from: —CH₂ NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂ SO—,by methods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may be used in certain embodimentsto generate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

“Antibody” or “antibody peptide(s)” both refer to an intact antibody, ora fragment thereof. In certain embodiments, the antibody fragment may bea binding fragment that competes with the intact antibody for specificbinding. The term “antibody” also encompasses polyclonal antibodies andmonoclonal antibodies. In certain embodiments, binding fragments areproduced by recombinant DNA techniques. In certain embodiments, bindingfragments are produced by enzymatic or chemical cleavage of intactantibodies. In certain embodiments, binding fragments are produced byrecombinant DNA techniques. Binding fragments include, but are notlimited to, Fab, Fab′, F(ab′)2, Fv, Facb, and single-chain antibodies.Non-antigen binding fragments include, but are not limited to, Fcfragments.

“Chimeric antibody” refers to an antibody that has an antibody variableregion of a first species fused to another molecule, for example, anantibody constant region of another second species. In certainembodiments, the first species may be different from the second species.In certain embodiments, the first species may be the same as the secondspecies. In certain embodiments, chimeric antibodies may be made throughmutagenesis or CDR grafting to match a portion of the known sequence ofan antibody variable region. CDR grafting typically involves graftingthe CDRs from an antibody with desired specificity onto the frameworkregions (FRs) of another antibody.

A bivalent antibody other than a “multispecific” or “multifunctional”antibody, in certain embodiments, typically is understood to have eachof its binding sites identical.

An antibody substantially inhibits adhesion of a ligand to a receptorwhen an excess of antibody reduces the quantity of receptor bound to theligand by at least about 20%, 40%, 60%, 80%, 85%, or more (as measuredin an in vitro competitive binding assay).

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. An antibody specifically binds an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. In certain embodiments, an antibodyspecifically binds an antigen when the dissociation constant is ≦1M, incertain embodiments, when the dissociation constant is ≦100 nM, and incertain embodiments, when the dissociation constant is ≦10 nM.

As used herein, the term “label” refers to any molecule that can bedetected. In a certain embodiment, a polypeptide may be labeled byincorporation of a radiolabeled amino acid. In a certain embodiment,biotin moieties that can be detected by marked avidin (e.g.,streptavidin containing a fluorescent marker or enzymatic activity thatcan be detected by optical or colorimetric methods) may be attached tothe polypeptide. In certain embodiments, a label may be incorporatedinto or attached to another reagent which in turn binds to the antibodyof interest. For example, a label may be incorporated into or attachedto a polypeptide that in turn specifically binds the polypeptide ofinterest. In certain embodiments, the label or marker can also betherapeutic. Various methods of labeling polypeptides and glycoproteinsare known in the art and may be used. Certain general classes of labelsinclude, but are not limited to, enzymatic, fluorescent,chemiluminescent, and radioactive labels. Examples of labels forpolypeptides include, but are not limited to, the following:radioisotopes or radionucleoides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., fluorescein isothocyanate(FITC), rhodamine, lanthanide phosphors, phycoerythrin (PE)), enzymaticlabels (e.g., horseradish peroxidase, β-galactosidase, luciferase,alkaline phosphatase, glucose oxidase, glucose-6-phosphatedehydrogenase, alcohol dehyrogenase, malate dehyrogenase,penicillinase), chemiluminescent, biotinyl groups, predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). In certain embodiments, labels areattached by spacer arms of various lengths to reduce potential sterichindrance.

The term “sample”, as used herein, includes, but is not limited to, anyquantity of a substance. In certain embodiments, a sample may be from achemical reaction, including, but not limited to, a protein synthesisreaction.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient.

The term “modulator,” as used herein, is a compound that changes oralters the activity or function of a molecule. For example, a modulatormay cause an increase or decrease in the magnitude of a certain activityor function of a molecule compared to the magnitude of the activity orfunction of the molecule in the absence of the modulator. In certainembodiments, a modulator is an inhibitor, which decreases the magnitudeof at least one activity or function of a molecule. Certain exemplaryactivities and functions of a molecule include, but are not limited to,binding affinity, enzymatic activity, and signal transduction. Certainexemplary inhibitors include, but are not limited to, proteins,peptides, antibodies, peptibodies, carbohydrates or small organicmolecules. Peptibodies are described, e.g., in WO 01/83525.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more

abundant than any other individual species in the composition). Incertain embodiments, a substantially purified fraction is a compositionwherein the object species comprises at least about 50 percent (on amolar basis) of all macromolecular species present. In certainembodiments, a substantially pure composition will comprise more thanabout 80%, 85%, 90%, 95%, or 99% of all macromolar species present inthe composition. In certain embodiments, the object species is purifiedto essential homogeneity (contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species.

The term “patient” includes human and animal subjects.

Certain Exemplary Response Element Regions and Reporter Nucleic AcidConstructs

As discussed above, a response element region refers to a region of adouble-stranded nucleic acid that is capable of being bound by one ormore activated response element transcription factors, or one or morecomplexes of activated response element transcription factors, tomodulate expression of one or more genes. A response element region alsorefers to a region of a single-stranded nucleic acid that, if it weredouble-stranded DNA, would be capable of being bound by one or moreactivated response element transcription factors, or one or morecomplexes of activated response element transcription factors, tomodulate expression of one or more genes. In this patent application,discussion of one or more transcription factor binding to a responseelement region encompasses binding by one or more transcription factorand/or binding by one or more complexes of activated transcriptionfactors. In certain embodiments, a response element region is provided.

In certain embodiments, a response element region is included in areporter nucleic acid construct, which is transfected into a signalingmolecule-responsive host cell. In certain embodiments, a reporternucleic acid construct comprises at least a response element region, apromoter, and a reporter nucleic acid in operable combination. Incertain embodiments, when a signaling molecule interacts with asignaling molecule receptor of the host cell, a process is triggeredthat results in activation of one or more response element transcriptionfactor. In certain embodiments, the activated one or more responseelement transcription factor binds to the response element region of thereporter nucleic acid construct, which results in expression of thereporter nucleic acid. In certain embodiments, one can then detect theproduction of the reporter polypeptide to determine the activity of thesignaling molecule.

Certain embodiments involve response element transcription factors thatare known as Signal Transducers and Activators of Transcription (STAT).At least seven members of the STAT family of proteins have beenidentified in mammals, including Stat1, Stat2, Stat3, Stat4, Stat5a,Stat5b, and Stat6. The term STAT5 encompasses both STAT5a or STAT5.Cytokine or growth factor binding to certain cell surface receptorsresults in tyrosine phosphorylation and activation of STATs in thecytoplasm. Phosphorylated STATs form dimers through reciprocalphosphotyrosine-SH2 interactions. The activated STAT dimers translocateto the nucleus, where they activate transcription of STAT-responsivegenes by binding to STAT-specific DNA response elements. See, e.g.,Turkson et al. (2000) Oncogene, 19: 6613 and references cited therein.

STAT proteins serve a diverse array of biological functions, including,but not limited to, roles in differentiation, proliferation,development, apoptosis, and inflammation. The important physiologicalrole of certain STATs has been demonstrated through various mouseknock-out experiments. For example, Stat2 null mice and Stat3 null miceare both embryonic lethal, while Stat1 null mice show highsusceptibility to certain infections, reduced interferon responses, andhigher incidence of certain tumors. Stat5a and/or Stat5b knockout miceare viable but show a variety of tissue-specific defects. See, e.g.,Turkson et al. (2000) Oncogene, 19: 6613 and references cited therein.

In certain embodiments, a response element region that can be bound bySTAT5 is provided. In certain embodiments, a response element regioncomprises (i) the sequence GTCATTTCCAGGAAATCACC or (ii) a sequencecomplementary to the sequence in (i).

In certain embodiments, the response element region comprises:

(a) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC or (ii) asequence complementary to the sequence in (i);

(b) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACCor (ii) a sequence complementary to the sequence in (i);

(c) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC or (ii) a sequence complementary to thesequence in (i); or

(d) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC or (ii) a sequence complementaryto the sequence in (i);

wherein Y, X, and Z are each independently selected from a nucleic acidsequence of 0 to 48 (including any integer within that range)nucleotides. In certain embodiments, Y, X, and Z are each independentlyselected from a nucleic acid sequence of 0 to 23 nucleotides.

In certain embodiments, Y, X, and/or Z may serve as spacer elements toprovide space between multiple triple repeat sequences. In certainembodiments, a spacer element is a multiple of 10 to 12 nucleotideslong, which results in the center regions of the sequencesGTCATTTCCAGGAAATCACC being spatially oriented to be in approximately thesame location (on the y and z axis) plus or minus 36 degrees, relativeto the center axis of the double-helical DNA (x-axis), wherein thecenter region is the tenth and eleventh nucleotides AG of the sequenceGTCATTTCCAGGAAATCACC. FIG. 1 is an exemplary representation of thesequence GTCATTTCCAGGAAATCACC on a double helix DNA. FIG. 1 also depictsthe x, y, and z axis.

In certain embodiments, a spacer element is 10 to 12 nucleotides long.In certain embodiments, a spacer element is 20 to 24 nucleotides long.In certain embodiments, a spacer element is 23 nucleotides long. Incertain embodiments, a spacer element is 30 to 36 nucleotides long. Incertain embodiments, a spacer element is 8 to 12 nucleotides long. Incertain embodiments, a spacer element is 16 to 24 nucleotides long. Incertain embodiments, a spacer element is 24 to 36 nucleotides long.

In certain embodiments, Y, X, and/or Z may serve a functional role intranscription. For example, in certain embodiments,

transcription factors may bind to Y, X, and/or Z. Exemplarytranscription factors that may bind may bind to Y, X, and/or Z, include,but are not limited to, —NFAT, AP-1, CRE, NFκB, and members of the STATprotein family.

In certain embodiments, an isolated nucleic acid comprises the sequence:GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC, wherein Y, X, and Z are each a nucleic acid sequence of 0nucleotides.

In certain embodiments, an isolated nucleic acid comprises the sequence:GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC, wherein Y, X, and Z are each a nucleic acid sequence of 8nucleotides. In certain embodiments, Y, X, and Z are each the nucleicacid sequence GCCGTACC.

In certain embodiments, an isolated nucleic acid comprises the sequence:GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC, wherein Y is a nucleic acid sequence of 8 nucleotides, X is anucleic acid sequence of 10 nucleotides, and Z is a nucleic acidsequence of 16 nucleotides. In certain embodiments, Y is the nucleicacid sequence GCCGTACC, X is the nucleic acid sequence TACCGGTCTG, and Zis the nucleic acid sequence ACCGGCCTAGTGCGTC.

In certain embodiments, an isolated nucleic acid comprises the sequence:

GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC,wherein Y and X are each a nucleic acid sequence of 0 nucleotides.

In certain embodiments, an isolated nucleic acid comprises the sequence:

GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC,wherein Y and X are each a nucleic acid sequence of 8 nucleotides. Incertain embodiments, Y and X are each the nucleic acid sequenceGCCGTACC.

In certain embodiments, an isolated nucleic acid comprises the sequence:

GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC,wherein Y is a nucleic acid sequence of 8 nucleotides and X is a nucleicacid sequence of 10 nucleotides. In certain embodiments, Y is thenucleic acid sequence GCCGTACC and X is the nucleic acid sequenceTACCGGTCTG.

In certain embodiments, a response element region comprises:

(i) the sequence N₅TTCCQGGAAN₆; wherein N₅ is a sequence of fivenucleotides independently selected from A, T, C, or G; N₆ is a sequenceof six nucleotides independently selected from A, T, C, or G; and Q isnucleotide A, C, or T; or

(ii) a sequence complementary to the sequence in (i). In certainembodiments, Q is the nucleotide A. In certain embodiments, Q is thenucleotide C. In certain embodiments, Q is the nucleotide T.

In certain embodiments, a response element region comprises:

(i) the sequence N₄TTTCCQGGAAAN₅; wherein N₄ is a sequence of fournucleotides independently selected from A, T, C, or G; N₅ is a sequenceof five nucleotides independently selected from A, T, C, or G; and Q isnucleotide A, C, or T; or

(ii) a sequence complementary to the sequence in (i). In certainembodiments, Q is the nucleotide A. In certain embodiments, Q is thenucleotide C. In certain embodiments, Q is the nucleotide T.

In certain embodiments, a response element region comprises:

(i) the sequence N₄TTTCCCCGAAAN₅; wherein N₄ is a sequence of fournucleotides independently selected from A, T, C, or G and N₅ is asequence of five nucleotides independently selected from A, T, C, or G;or

(ii) a sequence complementary to the sequence in (i).

In certain embodiments, a response element region comprises:

(i) the sequence N₄ATTCTCAGAAAN₅; wherein N₄ is a sequence of fournucleotides independently selected from A, T, C, or G and N₅ is asequence of five nucleotides independently selected from A, T, C, or G;or

(ii) a sequence complementary to the sequence in (i).

In certain embodiments, a response element region comprises:

(i) the sequence N₄TTTCTAGGAATN₅; wherein N₄ is a sequence of fournucleotides independently selected from A, T, C, or G and N₅ is asequence of five nucleotides independently selected from A, T, C, or G;or

(ii) a sequence complementary to the sequence in (i).

In certain embodiments, a response element region comprises:

(a) the sequence E₃ or a sequence complementary to the sequence E₃;

(b) the sequence E₃-X-E₃ or a sequence complementary to the sequenceE₃-X-E₃;

(c) the sequence E₃-X-E₃-Y-E₃ or a sequence complementary to thesequence E₃-X-E₃-Y-E₃; or

(d) the sequence E₃-X-E₃-Y-E₃-Z-E₃ or a sequence complementary to thesequence E₃-X-E₃-Y-E₃-Z-E₃;

wherein Y, X, and Z are each independently selected from a nucleic acidsequence of 0 to 48 (including any integer within that range)nucleotides, and wherein E₃ is [N₅TTCCQGGAAN₆]₃; [N₄TTTCCQGGAAAN₅]₃;[N₄TTTCCCCGAAAN₅]₃; [N₄ATTCTCAGAAAN₅]₃; or [N₄TTTCTAGGAATN₅]₃; wherein Qis the nucleotide A, C, or T; N₄ is a sequence of four nucleotidesindependently selected from A, T, C, or G; N₅ is a sequence of fivenucleotides independently selected from A, T, C, or G; and N₆ is asequence of six nucleotides independently selected from A, T, C, or G.

In certain embodiments, Y, X, and/or Z may serve as spacer elements toprovide space between multiple triple repeat sequences. In certainembodiments, a spacer element is a multiple of 10 to 12 nucleotideslong, which results in the center regions of each repeat sequence beingspatially oriented to be in approximately the same location (on the yand z axis) plus or minus 36 degrees, relative to the center axis of thedouble-helical DNA (x-axis), wherein the center region is the middle twonucleotides of the particularly recited sequence. In certainembodiments, a spacer element is 10 to 12 nucleotides long. In certainembodiments, a spacer element is 20 to 24 nucleotides long. In certainembodiments, a spacer element is 23 nucleotides long. In certainembodiments, a spacer element is 30 to 36 nucleotides long. In certainembodiments, a spacer element is 8 to 12 nucleotides long. In certainembodiments, a spacer element is 16 to 24 nucleotides long. In certainembodiments, a spacer element is 24 to 36 nucleotides long.

In certain embodiments, Y, X, and/or Z may serve a functional role intranscription. For example, in certain embodiments, transcriptionfactors may bind to Y, X, and/or Z. Exemplary transcription factors thatmay bind may bind to Y, X, and/or Z, include, but are not limited to,—NFAT, AP-1, CRE, NFκB, and members of the STAT protein family.

In certain embodiments, a response element region contains more than oneset of sequences that bind at least one STAT protein. In certain suchembodiments, each set of sequences that bind at least one STAT proteinis embedded in the center of a twenty (20)-mer, where the sequencesflanking the set of sequences that bind at least one STAT protein can beany nucleotides A, T, C, or G to complete the twenty-mer. Certain STATprotein binding sites are disclosed in PCT Publication No. WO 95/28482.

In certain embodiments, a reporter nucleic acid construct is provided.Various reporter nucleic acid constructs comprise various components inaddition to a response element region.

In certain embodiments, a response element region is operably linked toa promoter. In certain embodiments, a promoter is capable of being bounddirectly or indirectly by a polymerase, which results in transcriptionof a downstream encoding sequence. Many different promoters may be usedaccording to various embodiments. In various embodiments, promoters maybe eukaryotic, prokaryotic, or viral promoters that are capable ofdriving transcription of an encoding sequence when transfected into ahost cell.

One skilled in the art will be able to determine suitable promoters foruse in a given reporter nucleic acid construct and a given host cell.Nonlimiting exemplary promoters include, but are not limited to, SV40promoter, thymidine kinase (TK) promoter, PGK promoter, beta actinpromoter, CMV promoter, RSV promoter, MSCV promoter, MuLV promoter, HIVpromoter, and polyhedron promoter, and fragments and minimal promotersbased on any of these promoters. Nonlimiting exemplary promoters aredescribed, e.g., in PCT Publication No. WO 95/28482.

In certain embodiments, a reporter nucleic acid construct comprises areporter nucleic acid, which encodes a polypeptide that can be used todetect expression of the reporter nucleic acid. Many different reporternucleic acids may be used according to various embodiments. Thepolypeptide expressed by a reporter nucleic acid (reporter polypeptide)may be detected directly or indirectly.

In certain embodiments, a reporter polypeptide may be detected by usinga molecule that binds to the reporter polypeptide. In various

embodiments, the molecule that binds the reporter polypeptide may be anymolecule that has affinity for the reporter polypeptide. In certainembodiments, the molecule that binds the reporter polypeptide islabeled. In certain embodiments, the molecule that binds to the reporterpolypeptide is an antibody. In certain embodiments, a reporterpolypeptide may be detected by its interaction with another molecule. Incertain embodiments, the reporter polypeptide is an enzyme thatinteracts with another molecule to provide a signal.

Nonlimiting exemplary reporter polypeptides include, but are not limitedto, fluorescent molecules, chemilluminescent molecules,electrochemillunescent molecules, luciferase (LUC), phosphatase,alkaline phosphatase, placental alkaline phosphatase,beta-galactosidase, green fluorescent protein, beta-lactamase.Nonlimiting exemplary reporter polypeptides are described, e.g., in PCTPublication No. WO 95/28482.

In certain embodiments, a reporter nucleic acid construct is used totest the activity of a signaling molecule and/or the ability of a testcompound to affect the activity of a signaling molecule. As discussedabove, a signaling molecule refers to an extracellular molecule, ineither a free or bound form, that interacts with a receptor of a cell,which triggers a process that affects the activity of one or moreresponse element transcription factor. Many different signalingmolecules may be used according to various embodiments. In certainembodiments, a signaling molecule interacts with a receptor of a cell,which triggers a process that activates one or more response elementtranscription factor. In certain embodiments, a signaling moleculeinteracts with a receptor of a cell, which triggers a process thatdeactivates one or more response element transcription factor. Incertain embodiments, activation of one or more response elementtranscription factor results in binding of the one or more activatedresponse element transcription factor to a response element region,which results in expression of a reporter nucleic acid.

Exemplary signaling molecules include, but are not limited to,polypeptides, oligosaccarides, small organic molecules, antibodies,peptibodies, carbohydrates, peptide mimetics, fusion proteins, complexorganic molecules (including, but not limited to, steroids), and lipids(including, but not limited to, phospholipids). In certain embodiments,a signaling molecule is a cytokine. In certain embodiments, a signalingmolecule is a growth factor. Nonlimiting exemplary signaling moleculesare described, e.g., in PCT Publication No. WO 95/28482. In certainembodiments, more than one signaling molecule may be involved in aprocess of modulating response element transcription factor activity.

In certain embodiments, a signaling molecule interacts with a receptorof a cell, which triggers a process that results in activation of STATS.Exemplary signaling molecules that have been shown to be involved intriggering such a process in certain instances include, but are notlimited to, Interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9,IL-10, IL-12, IL-13, IL-15, IL-15, IL-27, erythropoietin, tPO, G-CSF,GM-CSF, growth hormone, TSLP, cKit, erythropoeitic products, GCSF-likemolecules, and prolactin. Exemplary signaling molecules include, but arenot limited to: naturally occurring polypeptides; polypeptides that havea naturally occurring amino acid sequence; polypeptides that have anamino acid sequence that is 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to a naturally occurring amino acid sequence; fusionproteins that comprise at least one naturally occurring amino acidsequence; chemically modified polypeptides; polypeptides with a polymerattached, e.g., polyethylene glycol (PEG); and molecules that have thesignaling molecule activity of a naturally occurring polypeptide.

The term “G-CSF” as used herein is defined as naturally occurring humanand heterologous species granulocyte colony-stimulating factor,recombinantly produced granulocyte colony-stimulating factor that is theexpression product consisting of either 174 or 177 amino acids, orfragments, analogs, variants, or derivatives thereof as reported, forexample in Kuga et al., Biochem. Biophys. Res. Comm. 159: 103-111(1989); Lu et al., Arch. Biochem. Biophys. 268: 81-92 (1989); U.S. Pat.Nos. 4,810,643, 4,904,584, 5,104,651, 5,214,132, 5,218,092, 5,362,853,5,606,024, 5,824,778, 5,824,784, 6,017,876, 6,166,183, and 6,261,550;U.S. Pat. Appl. No. US 2003/0064922; EP 0335423; EP 0 272703; EP 0459630; EP 0 256843; EP 0 243153; WO 9102874; Australian Applicationdocument Nos. AU-A-10948/92 and AU-A-76380/91. Included are chemicallymodified granulocyte colony-stimulating factors, see, e.g., thosereported in WO 9012874, EP 0401384 and EP 0335423. See also, WO03006501; WO 03030821; WO 0151510; WO 9611953; WO 9521629; WO 9420069;WO 9315211; WO 9305169; JP 04164098; WO 9206116; WO 9204455; EP 0473268;EP 0456200; WO 9111520; WO 9105798; WO 9006952; WO 8910932; WO 8905824;WO 9118911; and EP 0 370205. Also encompassed herein are all forms ofgranulocyte colony-stimulating factor, such as Albugranin™, Neulasta™,Neupogen®, and Granocyte®.

Derivatives of G-CSF include molecules modified by one or more watersoluble polymer molecules, such as polyethylene glycol, or by theaddition of polyamino acids, including fusion proteins (procedures forwhich are well-known in the art). Such derivatization may occursingularly at the N- or C-terminus or there may be multiple sites ofderivatization. Substitution of one or more amino acids with lysine mayprovide additional sites for derivatization. (See U.S. Pat. No.5,824,784 and U.S. Pat. No. 5,824,778, incorporated by referenceherein).

The term “G-CSF-like molecules” means a molecule that can activate theSTAT protein through the granulocyte colony-stimulating factor receptoror portions thereof.

As meant herein, the term “erythropoietic product” means a product thatcan activate the STAT protein through the erythropoietin receptor orportions thereof. An erythropoietic product comprises an “erythropoieticglycoprotein” (as defined herein), which glycoprotein can be conjugatedto a non-protein molecule. An erythropoietic product as used hereinincludes naturally occurring human and hereologous specieserythropoietin, recombinantly-produced erythropoietin, such as Epogen®(epoietin alfa), Aranesp® (darbepoetin alfa), biologically activeprecursors, mimetics, analogs, variants, or derivatives thereof asreported, for example in U.S. Pat. Nos. 6,586,398, 6,319,499, 5,955,422,5,856,298, 5,441,868, 4,703,308, WO 91/05867, WO 95/05465, and WO96/40749 (all incorporated by reference herein).

An erythropoietic glycoprotein is preferably a secreted, recombinantprotein. Included among these erythropoietic products are erythropoieticglycoproteins that have been chemically modified, for example anerythropoietin glycoprotein conjugated to polyethylene glycol, such asthose disclosed in International Application Nos. WO 01/02017 and WO01/76640, U.S. Pat. Nos. 6,077,939, 5,643,575, 6,340,742, US PatentApplication No. 2002/0115833, and European Patent No. 1 064 951 (allincluded by reference herein). Further, an erythropoietic product can bea composition that comprises an erythropoietic glycoprotein and,optionally, one or more additional components such as a physiologicallyacceptable carrier, excipient, or diluent. For example, a compositionmay comprise an erythropoietic glycoprotein as described herein plus abuffer, an antioxidant such as ascorbic acid, a low molecular weightpolypeptide (such as those having less than 10 amino acids), a protein,amino acids, carbohydrates such as glucose, sucrose, or dextrins,chelating agent such as EDTA, glutathione, and/or other stabilizers,excipients, and/or preservatives. The composition may be formulated as aliquid or a lyophilizate. Further examples of components that may beemployed in pharmaceutical formulations are presented in Remington'sPharmaceutical Sciences, 16^(th) Ed., Mack Publishing Company, Easton,Pa., (1980). Further, the term “erythropoietic products” includessolutions or formulations comprising the erythropoietic glycoproteinsdescribed below, which may have enhanced stability and/or activity, suchas those described in U.S. Pat. Nos. 4,806,524, 6,333,306, and 6,277,367(all incorporated by reference herein).

As meant herein, the term “erythropoietic glycoprotein” encompassesglycoproteins that have the same amino acid sequence as any mammalianerythropoietin glycoprotein, including human erythropoietin, as well asanalogs and variants of each, i.e., erythropoietic glycoprotein analogsand erythropoietic glycoprotein variants. Human erythropoietin sequencesare disclosed in U.S. Pat. Nos. 4,703,008 and 5,688,679, European PatentNo. 0 205 564, and International Application No. WO 02/085940 (allincorporated herein by reference). Primate erythropoietin sequences aredisclosed in, e.g., U.S. Pat. Nos. 4,703,008 and 6,555,343 and NationalCenter for Biotechnology Information (NCBI) accession no. AAA36842, andother mammalian erythropoietins are disclosed in, for example,International Application No WO 99/54486 (canine erythropoietin), NCBIaccession nos. AAA37570 (mouse erythropoietin), AAA30842 (canineerythropoietin), AAA30807 (cat erythropoietin), and AAA31029 (pigerythropoietin), among many other published mammalian erythropoietinsequences (all incorporated herein by reference). Erythropoieticglycoproteins produced and/or purified by methods described in U.S. Pat.Nos. 4,667,016, 6,399,333, 6,391,633, and 6,355,241 (all incorporatedherein by reference) are erythropoietic glycoproteins as meant herein.

Specifically included within “erythropoietic glycoprotein analogs” areglycoproteins that are substantially similar to a mammalianerythropoietin and that can stimulate erythropoiesis as demonstrated inan in vivo bioassay, for example, the exhypoxic polycythemic mouseassay. See e.g. Cotes and Bangham (1961), Nature 191: 1065. Examples oferythropoietic glycoprotein analogs include the analogs described inInternational Application Nos. WO 95/05465 and WO 01/81405 (incorporatedherein by reference) which provide analogs of human erythropoietincomprising more N-glycan sites than are present in unaltered humanerythropoietin. For example, the analog designated N47 in WO 95/05465comprises five N-glycan sites, and the analog designated N66 in WO01/81405 comprises seven N-glycan sites rather than the three present inunaltered human erythropoietin. Other erythropoietic glycoproteinanalogs include those comprising any single alteration described inInternational Application Nos. WO 95/05465 and/or WO 01/81405 or anycombination of such alterations, provided that the resulting protein isstill substantially similar to human erythropoietin. Other examplesinclude the erythropoietic glycoprotein analogs disclosed in U.S. Pat.Nos. 5,856,298 and 6,153,407, International Application Nos. WO00/24893, WO 91/05867, and WO 00/24893, and WO 03/029291, and EuropeanPatent Application 0 902 085 (all herein incorporated by reference). Onesuch erythropoietic glycoprotein analog has been given the United StatesAdopted Name (USAN) darbepoetin alfa and is marketed under the tradenameARANESP® by Amgen Corporation of Thousand Oaks, Calif., USA. Still othererythropoietic glycoprotein analogs include human erythropoietin with anadditional amino acid at the carboxy-terminus, for example arginine.

Included among “erythropoietic glycoprotein variants” are moleculescomprising an erythropoietic glycoprotein analog, as defined above, amammalian erythropoietin, or a fragment either of these that canstimulate erythropoiesis fused to a different protein, polypeptide, orfragment thereof. Erythropoietin variants include the fusion proteinsdescribed in, e.g., U.S. Pat. No. 6,548,653, among many others such asvariants including the Fc region of an antibody or a dimerization ortrimerization domain, for example a leucine zipper.

Human erythropoietin is 165 amino acids long and has three N-glycansites that allow attachment of N-glycans at amino acids 24, 38, and 83and one O-glycan site that allows attachment of an O-glycan at aminoacid 126. This protein is described in U.S. Pat. No. 4,703,008, where itis described by amino acids 1 to 165 in FIG. 6. An analog of humanerythropoietin, N47, which is produced by cells used in Examples 1, 2,and 4, is also 165 amino acids long and has five N-glycan sites thatallow attachment of N-glycans at amino acids 24, 30, 38, 83, and 88 andone O-glycan site that allows attachment of an O-glycan at amino acid126. This protein is described in International Application No. WO95/05465, where it is designated analog “N47.”

In certain embodiments, a signaling molecule interacts with a receptorof a cell, which triggers a process that results in activation of one ormore STAT protein other than STATS. In certain embodiments, a signalingmolecule interacts with a receptor of a cell, which triggers a processthat results in activation of one or more response element transcriptionfactor other than a STAT protein.

In certain embodiments, a signaling molecule-responsive host cell isprovided. Many different signaling molecule-responsive host cells may beused according to various embodiments. As discussed above, a signalingmolecule-responsive host cell refers to a host cell that comprises asignaling molecule receptor cabable of interacting with a signalingmolecule. In certain embodiments, a signaling molecule receptor iscapable of interacting with a signaling molecule described above. Incertain embodiments, a signaling molecule receptor is a cytokinereceptor. In certain embodiments, a signaling molecule receptor is agrowth factor receptor. Nonlimiting exemplary signaling moleculereceptors are described, e.g., in PCT Publication No. WO 95/28482.

In certain embodiments, a signaling molecule receptor is capable ofinteracting with a signaling molecule, which triggers a process thatresults in activation of STATS. Exemplary signaling molecule receptorsinvolved in triggering such a process in certain instances include, butare not limited to, Interleukin (IL)-1 receptor, IL-2 receptor, IL-3receptor, IL-4 receptor, IL-5 receptor, IL-7 receptor, IL-9 receptor,IL-10 receptor, IL-12 receptor, IL-13 receptor, IL-15 receptor, IL-15receptor, IL-27 receptor, Epo receptor, tPO receptor, G-CSF receptor,GM-CSF receptor, growth hormone receptor, TSLP receptor, cKit receptor,and prolactin receptor.

In certain embodiments, a signaling molecule receptor is capable ofinteracting with a signaling molecule, which triggers a process thatresults in activation of one or more STAT protein other than STAT5. Incertain embodiments, a signaling molecule receptor is capable ofinteracting with a signaling molecule, which triggers a process thatresults in activation of one or more response element transcriptionfactor other than a STAT protein.

In certain embodiments, a signaling molecule receptor is a chimericsignaling molecule receptor. In certain embodiments, a chimericsignaling molecule receptor comprises a molecule comprising at least twoportions from different receptor molecules. A nonlimiting exemplarychimeric signaling molecule receptor molecule is a molecule comprisingat least a portion of the extracellular domain of the Epo receptor andat least a portion of the cytoplasmic domain of prolactin receptor. See,e.g., Socolovsky et al., Journal of Biological Chemistry,272(22):14009-14012 (1997).

In certain embodiments, a signaling molecule-responsive host cellexpresses a signally molecule receptor from an endogenous gene. Incertain embodiments, a signaling molecule-responsive host cell expressesfrom an endogenous gene one or more response element transcriptionfactor, whose activity is affected by the interaction of a signalingmolecule receptor and a signaling molecule. In certain embodiments, asignaling molecule-responsive host cell expresses a signaling moleculereceptor from an endogenous gene, and expresses from an endogenous geneone or more response element transcription factor, whose activity isaffected by the interaction of the signaling molecule receptor and asignaling molecule. This discussion of expressing a signaling moleculereceptor encompasses host cells that express more than one type ofsignaling molecule receptor.

In certain embodiments, a signaling molecule-responsive host cellexpresses one or more signaling molecule receptors from one or moreexogenous nucleic acids. In certain embodiments, the signalingmolecule-responsive host cell expresses one or more response elementtranscription factors from one or more exogenous nucleic acids. Incertain embodiments, one or more exogenous nucleic acids have beentransiently introduced into the host cell. In certain embodiments, oneor more exogenous nucleic acids have been stably introduced into thehost cell. In various embodiments, a nucleic acid that can express asignaling molecule receptor and can express one or more response elementtranscription factor has been introduced into a signalingmolecule-responsive host cell by any method known in the art. In certainembodiments, a nucleic acid that can express a signaling moleculereceptor and a separate nucleic acid that can express one or moreresponse element transcription factor have been introduced into asignaling molecule-responsive host cell.

In certain embodiments, a signaling molecule-responsive host cell mayexpress a signaling molecule receptor from an endogenous gene, but toincrease the amount of receptor, the host cell also expresses thesignaling molecule receptor from an exogenous nucleic acid. In certainembodiments, a host cell may not express a signaling molecule receptorfrom an endogenous gene, and the host cell expresses the signalingmolecule receptor from an exogenous nucleic acid. This discussion of ahost cell that expresses a signaling molecule receptor encompasses hostcells that express more than one type of signaling molecule receptorfrom an exogenous nucleic acid.

In certain embodiments, a host cell may express one or more responseelement transcription factors from an endogenous gene, but to increasethe amount one or more response element transcription factor, the hostcell also expresses one or more response element transcription factorfrom an exogenous nucleic acid. In certain embodiments, a host cell maynot express one or more response element transcription factor from anendogenous gene, and the host cell expresses one or more responseelement transcription factor from an exogenous nucleic acid. Thisdiscussion of a host cell that expresses one or more response elementtranscription factor encompasses host cells that express one or moreresponse element transcription factor from more than one exogenousnucleic acid.

Exemplary host cells that may transfected with a reporter nucleic acidconstruct include, but are not limited to, NIH-3T3 fibroblast cells, UT7cells, BaF3 cells, 32D clone 3 cells, 32D clone 23 cells, DA-1 cells,DA-3 cells, A431 cells, C3H10T1/2 cells, CaCo 2 cells, CHO cells, COS-7cells, CV-1 cells, Daudi cells, Jurkat cells, EL-4 cells, Hela cells,HEK293 cells, HUVEC cells, HL-60 cells, U937 cells, HepG2 cells, HT1080cells, HUT78 cells, L cells, MC/9 cells, RBL-1 cells, MO7e cells, Neuro2A cells, PC-12 cells, RAJI cells, Ramos cells, Rat-1 cells, Saos-2cells, ST-2 cells, THP-1 cells, TF1 cells, Wehi-3 cells, bone marrowcells, CD34 positive cells, embryonic stem cells, and germ cells.Nonlimiting exemplary host cells are described, e.g., in PCT PublicationNo. WO 95/28482.

UT7/Epo cells naturally comprise Epo receptors. Accordingly, in certainembodiments, UT7/Epo cells transfected with a reporter nucleic acidconstruct may be used to test Epo activity. BaF3 cells naturallycomprise IL-3 receptors. Accordingly, in certain embodiments, BaF3 cellstransfected with a reporter nucleic acid construct may be used to testIL-3 activity.

In certain embodiments, 32D clone 3 cells are transfected with a nucleicacid that expresses human G-CSF receptor and with a reporter nucleicacid construct. Accordingly, in certain embodiments, such transfectedcells can be used to test G-CSF activity. In certain embodiments,NIH-3T3 cells are transfected with a nucleic acid that expresses Eporeceptor and with a reporter nucleic acid construct. Accordingly, incertain embodiments, such transfected cells can be used to test Epoactivity.

Certain Exemplary Methods

In certain embodiments, a signaling molecule-responsive host cellcomprising a reporter nucleic acid construct may be used to determinethe activity of a test composition comprising a signaling molecule. Incertain embodiments, the signaling molecule-responsive host cellcomprising a reporter nucleic acid construct is contacted with the testcomposition under conditions in which the reporter nucleic acidconstruct expresses a reporter polypeptide in response to the signalingmolecule. If the test composition contains a sufficient amount of theactive signaling molecule, reporter polypeptide will be produced at alevel that can be detected.

As a non-limiting example, a signaling molecule-responsive host cell maybe transformed or transfected, either stably or transiently, with areporter nucleic acid construct that comprises at least a responseelement region, a promoter, and a reporter nucleic acid in operablecombination. If the reporter nucleic acid is a luciferase gene, forexample, then contacting the signaling molecule-responsive host cellcomprising the reporter nucleic acid construct with a test compositioncomprising the signaling molecule will result in expression ofluciferase, which may be detected using an appropriate assay and aluminometer. The amount of light produced may be directly related to theamount of luciferase protein expressed from the reporter nucleic acidconstruct, which in turn may be related to the amount of signalingmolecule activity present in the test composition.

In certain embodiments, the test composition comprising a signalingmolecule is a particular production batch of the signaling molecule. Incertain embodiments, a production batch of the signaling molecule isproduced by expressing a recombinant gene for the signaling molecule inprokaryotic or eukaryotic cells. In certain embodiments, the signalingmolecule is further purified to produce a production batch. In certainembodiments, a production batch of the signaling molecule is produced bypurifying the signaling molecule from cells that express the signalingmolecule from an endogenous gene. A production batch includes batchessuitable for experimental purposes as well as batches suitable forlarge-scale production of pharmaceutical molecules. Thus, a productionbatch may be of any size.

In certain embodiments, the test composition may be a compositioncomprising a sample suspected of containing the signaling molecule.Non-limiting exemplary samples include mammalian tissues and mammaliansamples, including, but not limited to, blood, urine, serum, saliva,muscle, bone bone marrow, thymus, spleen, kidney, liver, adrenal gland,brain, spinal fluid, peritoneal fluid, and bronchial lavage.Non-limiting exemplary samples also include, but are not limited to,plant tissues and cells, non-mammalian eukaryotic tissues and cells,prokaryotic cells, medium that has been in contact with cells, and anyof the above sources upon introduction of specific conditions, e.g.,varying nutrients and/or stress levels. In certain embodiments, thesample is from a nonbiological origin, for example from a chemicalsynthesis. Samples may be manipulated as appropriate, e.g., byextraction, solubilization, filtration, dilution, etc., in order to forma test composition. One skilled in the art can form an appropriate testcomposition from a particular sample.

The activity of the test composition comprising a signaling moleculemay, in certain embodiments, be compared to the activity of a standardcomposition comprising the signaling molecule subjected to the sameassay. A standard composition comprising the signaling molecule may, incertain embodiments, comprise a known concentration of the signalingmolecule (e.g., the standard composition may comprise x mg/mL or ymmol/mL of the signaling molecule). In certain embodiments, a standardcomposition comprising the signaling molecule may comprise a knownconcentration of signaling molecule activity (e.g., the standardcomposition may comprise z units of activity/mL). In certainembodiments, a standard composition comprising the signaling moleculemay comprise a known concentration of the signaling molecule and a knownconcentration of signaling molecule activity (e.g., the standardcomposition may comprise x mg/mL of signaling molecule and z units ofactivity/mL of signaling molecule activity, which means that thesignaling molecule in the standard composition has z units of activityper x mg of signaling molecule).

In certain embodiments, by comparing the activity of a test compositioncomprising a signaling molecule to a standard composition comprising thesignaling molecule, one skilled in the art may adjust the concentrationof the signaling molecule in the test composition or the concentrationof signaling molecule activity in the test composition as desired. Incertain embodiments, the concentration of the signaling molecule in thetest composition or the concentration of the signaling molecule activityin the test composition is adjusted to be the same as that of thestandard composition.

In certain embodiments, the activity of the test composition comprisinga signaling molecule may be compared to the activity of a blankcomposition that lacks the signaling molecule subjected to the sameassay. The activity of the test composition comprising the signalingmolecule may, in certain embodiments, be expressed as a“fold-stimulation” over the activity of the blank composition.Fold-stimulation may be calculated, in certain embodiments, by dividingthe signal of the test composition by the signal of the blankcomposition in the same assay. In certain embodiments, a backgroundsignal is subtracted from the signal of each test composition beforecalculating the fold-stimulation. In various embodiments, the backgroundsignal may be, e.g., the signal produced by the assay reagents withoutany cells, the signal produced by the signaling molecule-responsivecells lacking the reporter nucleic acid construct, or any otherappropriate background measure.

In certain embodiments, a signaling molecule-responsive host cellcomprising a reporter nucleic acid construct may be used to determinewhether a test compound has the activity of a particular signalingmolecule. In certain embodiments, the signaling molecule-responsive hostcell comprising a reporter nucleic acid construct is contacted with thetest compound, in the absence of the signaling molecule, underconditions in which the reporter nucleic acid construct expresses areporter protein in response to the signaling molecule. If the testcompound has the activity of the signaling molecule, reporter proteinwill be produced at a level that can be detected.

In certain embodiments, a test compound that has the activity of aparticular signaling molecule is referred to as an “agonist.” Agonistsinclude any molecule that results in the specific downstream effectsthat are characteristic of a particular signaling molecule. Thus, asused herein, an agonist is any molecule that, when contacted with acertain signaling molecule-responsive host cell comprising a certainreporter nucleic acid construct, results in expression of the reporterpolypeptide. An agonist need not function by the same mechanism as thesignaling molecule. Thus, as a non-limiting example, an agonist need notbind the signaling molecule's cognate receptor in the same location ormanner as the signaling molecule, and indeed, the agonist need not bindthe receptor at all.

In certain embodiments, the activity of a test compound that may havethe activity of a particular signaling molecule is compared to theactivity of a standard composition comprising the signaling molecule.One skilled in the art can select a test compound that has the desiredlevel of activity relative to a standard composition.

In certain embodiments, the activity of a test compound that may havethe activity of a particular signaling molecule is compared to theactivity of a blank composition that lacks the signaling molecule. Incertain embodiments, the activity of the test compound may be expressedas a fold-stimulation over the blank composition, substantially asdiscussed above.

In certain embodiments, a library of test compounds may be tested toselect compounds having the activity of a particular signaling molecule.In certain embodiments, the assays discussed herein may be adapted bymethods known in the art for high-throughput screening of a librarycomprising a large number of such test compounds.

In certain embodiments, a signaling molecule-responsive host cellcomprising a reporter nucleic acid construct may be used to determinewhether a test compound impacts the activity of a signaling molecule. Incertain embodiments, the signaling molecule-responsive host cellcomprising a reporter nucleic acid construct is contacted with thesignaling molecule and the test compound under conditions in which thereporter nucleic acid construct expresses a reporter protein in responseto the signaling molecule. In certain embodiments, the activity of thesignaling molecule alone is compared to the activity of the signalingmolecule in the presence of the test compound under the same assayconditions. If the test compound impacts the activity of the signalingmolecule, the level of reporter protein produced will be different fromthe level of reporter protein produced in the presence of the signalingmolecule alone.

In certain embodiments, a test compound that specifically inhibits theactivity of a signaling molecule is referred to as an “inhibitor.” Thus,an inhibitor includes any molecule that specifically reduces the levelof reporter protein expressed in response to the signaling molecule. Theinhibitor's mechanism of action is not limited. Thus, an inhibitor mayreduce the activity of a signaling molecule by any mechanism, including,but not limited to, preventing or reducing signaling molecule binding toits cognate receptor, preventing or reducing phosphorylation of anyprotein or proteins in the signaling pathway, and/or preventing orreducing activated response element transcription factor binding to thereporter nucleic acid construct.

In certain embodiments, a test compound may specifically increase theactivity of a signaling molecule. The mechanism of such an increase isnot limited. Thus, a test compound that specifically increases theactivity of a signaling molecule may function by any mechanism,including, but not limited to, mimicking the binding of the signalingmolecule to its cognate receptor, increasing and/or stabilizing thebinding of the signaling molecule to its cognate receptor, increasingthe extent and/or rate of phosphorylation of any protein or proteins inthe signaling pathway, and/or increasing the level and/or stability ofactivated response element transcription factor binding to the reporternucleic acid construct.

In certain embodiments, a response element region may be operably linkedto a promoter, which is operably linked to a gene of interest. A nucleicacid comprising the response element region operably linked to apromoter, which is operably linked to a gene of interest is referred toherein as an inducible nucleic acid construct. In certain embodiments,incubation of a host cell comprising an inducible nucleic acid constructwith a signaling molecule results in expression of the gene of interest.In that manner, in certain embodiments, expression of the gene ofinterest may be controlled by varying the concentration of signalingmolecule in contact with the host cell. In certain embodiments, cellscomprising the inducible nucleic acid construct may be grown to adesired density in the absence of the signaling molecule, and thensignaling molecule may be added to induce expression of the gene ofinterest. Such a system may be useful, in certain embodiments, forproducing a polypeptide product of a gene of interest, where thatpolypeptide product reduces the growth and/or is toxic to the hostcells. Such a system may also be useful, in certain embodiments, whenthe gene of interest is be activated, e.g., at a certain time point, ata certain cell density, or after certain other events have occurred.

Methods of producing proteins or polypeptides are known to one of skillin the art. One example is the production of erythropoietin as describedin U.S. Pat. No. 5,441,868. One of skill in the art would know how toproduce proteins from bacterial, mammalian systems in vivo, ex vivo, orin vitro.

EXAMPLES Example 1 Making pGLGTPLAP

A double-stranded nucleic acid having the following nucleic acidsequence was cloned into pBlueScript (Stratagene) cut with Asp718+SacIIto yield pBlue NS:

5′      CGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA  3′CAT GGCAGTCCCGGTCCTTTAGT GGCAGTAAAGGTCCTTTAGT   CCGTCATTTCCAG GAAATCA CCGC 3′    GGCAGTAAAGGTC CTTTAGT GG   5′Both strands are shown, including the overhangs generated for cloning.Bold indicates a response element repeat sequence. A double-strandednucleic acid having the following nucleic acid sequence was cloned intopBlueScript cut with SacII+HindIII to yield pBlue H/S:

5′    GGTCCCAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACACC A 3′ 3′C GCCAGGGTCCAGGTGAAGCGTATAATTCCACTGCGCACACCGGAGCTTGTGG TTCGA 5′Both strands are shown. including the overhangs generated for cloning.Underlining indicates a TK promoter sequence.

The Asp718-Sacll and SacII-HindIII fragments were released from pBlueA/S and pBlue H/S, respectively, and were cloned into Asp718+HindIII cutpGL3Basic (Promega) in a three-part ligation to yield pGTbasic, whichcomprised the following sequence:

GTA CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGCGGTCCCAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACA CC AAGCT(Only one strand is shown. Itallicized sequences indicate parts of therestriction sites used for cloning. Bold indicates a response elementrepeat sequence and underlining indicates a TK promoter sequence).

Construct pGLGFP was made by ligating the 250 base-pair NotI (filledin)-NcoI fragment from pGL3Basic into BglII cut (filled in) pEGFP(Clontech).

The 120 base-pair Asp718-HindIII (1×GAS-TKpromoter) fragment wasreleased from pGTbasic (discussed above) and ligated into Asp718-HindIIIcut pGLGFP to yield pGLGTGFP.

PLAP-76-SEQSIG comprises a human placental alkaline phosphatase (PLAP)cDNA with an artificially constructed N-terminus, including a secretionsignal sequence, and lacking the C-terminal glycosylphosphatidylinositol (GPI) anchor sequence. Specifically, the PLAP sequence inPLAP-76-SEQSIG comprises the following 5′ nucleotide sequence, whichincludes the artificially constructed N-terminus fused to nucleotides102 to 1571 of the PLAP sequence shown in Genbank #M13077:

5′ T CTAGACTCGA CATGCTGGGG CCCTGCATGC TGCTGCTGCT   GCTGCTGCTG GGCCTGAGGC TACAGCTCTC CCTGGGCATC    ATCGCGGCCG CAGGCATCAT 3′Only one strand is shown. The XbaI sequence at the 5′ end of the regionshown is underlined. The nucleotide sequence encoding theartificially-constructed N-terminus, including a secretion signalsequence, is in bold. Part of the native PLAP sequence beginning atnucleotide 102 of Genbank #M13077 is shown in italics. At the 3′ end ofthe PLAP insert in PLAP-76-SEQSIG is the following sequence:

5′ GCGCACCCGG GGGC TAGCTA AGGTACC 3′Only one strand is shown. The italicized portion is part of the nativePLAP sequence to nucleotide 1571 of Genbank #M13077. The stop codon isshown in bold and the Asp718 site is underlined.

Blunt-ended 1.5 Kb XbaI-Asp718 fragment from PLAP-76-SEQSIG, whichincludes the artificially constructed N-terminal signal sequence,nucleotides 102-1571 of human PLAP (Genbank #M13077), and the stopcodon, was ligated into blunt-ended HindIII-NotI cut pGLGTGFP to yieldpGLGTPLAP. Self-annealed double stranded nucleic acid 1596-13 (CGTACGGC)was ligated into SacII-cut pGTbasic to create a BsiWI site and yieldp1596-13/GTbasic comprising the following sequence:

GTA CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCA CCG ccgtacggCGGTCCCAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCG AACACC AAGCT(Only one strand is shown. Bold indicates a response element repeatsequence, underlining indicates a TK promoter sequence, and the lowercase sequence indicates nucleotides added by nucleic acid 1596-13).

The Asp718-BsiWI fragment from p1596-13/GTbasic was released and ligatedinto Asp718 cut pGLGTPLAP to generate a multi-GT-PLAP mixture (3Xresponse element repeat sequence; 6X response element repeat sequence,9X response element repeat sequence, 12X response element repeatsequence, >12X response element repeat sequence).

For example, a 9X Multi-GT-PLAP, comprises the following sequence:

1 GCAAGTGCAGGTGCCAGAACATTTCTCTATCGATAGGTA CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCA CCG ccgta CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGccgta CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCACCGTC ATTTCCAGGAAATCA CCG CGGTCCCAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACACC AAGCT CTAGACTCGACATGCTGGGGCCCTGCATG

Example 2 Testing with EPO

pGTPLAP DNAs with three (9X response element repeat sequence) or four(12X response element repeat sequence) copies of the response elementtriple repeat sequence were individually purified and linearized withApaL1 for transient transfection into NIH-3T3 fibroblasts (3×10e5cells/well) following standard procedures using Superfect (Qiagen) astransfection reagent and DMEM basal medium. The pGTPLAP constructs weretransfected into NIH-3T3 fibroblast cells alone or in variouscombinations with an expression plasmid for the Epo Receptor and/or anexpression plasmid for STAT5b (Azam et al, EMBO J, 14(7), 1402-1411,1995). The expression plasmid for the Epo Receptor was an MSCVretroviral expression vector into which human full length Epo Receptorhad been cloned. See Hawley et al., Gene Therapy, 1:136-138 (1994) forthe MSCV retroviral expression vecor and GenBank Accession No. 60459 forhuman Epo Receptor. Also, NIH-3T3 fibroblast cells were transfected withonly the expression plasmid for the Epo Receptor. Also, NIH-3T3fibroblast cells were transfected with only the expression plasmid forSTAT5b. Also, nontransfected NIH-3T3 fibroblast cells were used. Table 1below shows the various combinations that were tested. After a 2-3 hourincubation with the DNA-transfection mix, cells were washed with PBS andplaced in fresh 3T3 growth medium (phenol red-free DMEM, 10% FBS, 1XGlutamine).

After overnight recovery, medium was replaced with fresh 3T3 growthmedium with or without addition of 19 Units/ml of recombinant humanerythropoietin (rHuEpo) and left overnight again. Culture mediumsupernatants were collected and cleared of cells and debris either bycentrifugation at 15K rpm for 10 minutes (without filtration) or byfiltration (0.45 micron spin filter, 10 minutes, 15K rpm). Clearedsupernatants were heated at 65° C. for 1 hour and mixed with an equalvolume of 2× phosphatase reaction buffer containing 2M diethanolamine, 1mM MgCl₂, 20 mM homoarginine, 1 mg/ml BSA (pre-heated by itself for 1hour at 65° C. and then added), and 0.2 mM 4-methylumbelliferylphosphate dicyclohexylammonium trihydrate in water. Reactions wereallowed to proceed overnight at 37° C. and production of4-methylumbelliferyl as a measure of phosphatase activity was measuredat 360 nm/460 nm (excitation/emission wavelength). Results are shown inTable 1 below.

TABLE 1 fold −EPO +EPO stimulati

average − average − (avg +EPO −filtration +filtration baseline¹−filtration +filtration baseline avg −EP

1 9X-RS-pGTPLAP 30 24 12 30 26 13 1.0 2 9X-RS-pGTPLAP/EPOR 24 23 9 33 3519 2.0 3 9X-RS- 69 62 51 169 154 147 2.9 pGTPLAP/EPOR/STAT5b 412X-RS-Pgtplap 34 34 19 47 46 32 1.7 5 12X-RS-pGTPLAP/EPOR 32 32 17 4542 29 1.7 6 12X-RS- 110 102 91 200 183 177 2.0 pGTPLAP/EPOR/STAT5b 7EPOR 15 16 16 14 8 STAT5b 17 16 14 14 9 nontransfected NIH3T3 13 14 1717 ¹“average − baseline” equals the average (−filtration and+filtration) in the presence of a pGTPLAP less the approximate averageof all samples in the absence of a pGTPLAP. Thus, the baseline used forboth +EPO and −EPO was 15. RS = response element repeat sequence

indicates data missing or illegible when filed

Example 3 Testing with IL-3

pGTPLAP DNAs with increasing copy numbers of response element triplerepeat sequence were individually purified and linearized with ApaL1 forelectroporation (263-269 V/12.8-17.6 microF pulse; BTX Electro CellManipulator 600) into BaF3 cells which express endogenous IL-3receptors. Stable transfectants were selected for the presence of theNeomycin resistance gene (present in pGTPLAP) by a 2-week culture instandard culture medium (IMDM, 10% FetalClonell, 5×10e-5Mbeta-mercaptoethanol, 1X Glutamine, 2.5 ng/ml rMuIL-3 (Biosource))supplemented with G418 (750 microgram/ml).

To measure cytokine-induced PLAP expression, cells were washed andre-plated in phenolred-free RPMI containing 0.2% BSA, 5×10e-5Mbeta-mercaptoethanol, 1X Glutamine at 10e6 cells/0.5 ml/well andincubated overnight at 37° C. Also, nontransfected BaF3 cells were used.One of every two wells per construct was supplemented with 25 ng/mlrecombinant murine interleukin-3 (rMuIL-3) during the overnightincubation. Cell viability was assessed by Trypan Blue exclusion andculture medium supernatants were collected and cleared of cells anddebris either by centrifugation at 15K rpm for 10 minutes (withoutfiltration) or by filtration (0.45 micron spin filter, 10 minutes, 15Krpm). Cleared supernatants were heated at 65° C. for 1 hour and mixedwith an equal volume of 2× phosphatase reaction buffer containing 2Mdiethanolamine, 1 mM MgCL2, 20 mM homoarginine, 1 mg/ml BSA (pre-heatedby itself for 1 hour at 65° C. and then added), and 0.2 mM4-methylumbelliferyl

phosphate dicyclohexylammonium trihydrate (4-MUP) in water. Reactionswere allowed to proceed overnight at 37° C. and production of 4-MU as ameasure of phosphatase activity was measured at 360 nM/460 nM(excitation/emission wavelength). The results are shown in Table 2below.

TABLE 2 fold −IL-3 +IL-3 stimulation average − average − (avg +IL-3/−filtration +Filtration baseline¹ −filtration +Filtration baseline avg−IL-3) nontransfected 16 14 17 15 BaF3 cells 3X-RS- 35 33 20 85 82 703.5 pGTPLAP 6X-RS- 44 45 30 175 177 160 5.4 pGTPLAP 9X-RS- 53 54 40 276257 250 6.2 pGTPLAP 12X-RS- 59 63 45 402 384 375 8.4 pGTPLAP Greaterthan 48 48 33 384 389 370 11.2 12X-RS- pGTPLAP ¹“average − baseline”equals the average (−filtration and +filtration) in the presence of apGTPLAP less the approximate average (−filtration and +filtration) forall untransfected BaF3 cell samples. Thus, the baseline used for both+IL-3 and −IL-3 was 15. (The average baseline numbers in this table arerounded.) RS = response element triple repeat sequence

Example 4 Making HuG-CSFR-Iuc Cell Line

To generate the reporter nucleic acid construct, a DNA fragmentcontaining the 9X response element repeat sequence and the thymidinekinase promoter was removed from pGTPLAP by digesting with KpnI andXbaI. After removal, a fragment with the following sequence (thecomplementary strand will have both 5′ and 3′ overhangs resulting fromthe digestion):

CGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCG CCGTACCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGCCGTACCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGCGGTCCCAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACA CC AAGCTwas cloned into plasmid pGL3 basic (Promega) cut with KpnI and NheI(XbaI and NheI have compatible ends) to produce pGL3 basic containing a9X response element repeat sequence and the TK promoter.

pGL3 hygro was produced by cloning a hygromycin B resistance gene intothe BamHI site of the pGL3-promoter vector (Promega). A DNA fragmentcontaining the 9X response element repeat sequence and TK promoter wasreleased from the pGL basic3 vector by digestion with KpnI and HindIII.The DNA fragment was then ligated into pGL3 hygro cut with KpnI andHindIII to produce the reporter nucleic acid construct.

To generate the pool population of HuG-CSFR-luc cells, a clonalpopulation of 32D HuG-CSFR ck3 cells was used. This cell line expressesthe full-length human G-CSF receptor. The parental cell line 32D cl3 isnow available at ATCC (ATCC No. CRL-11346, ATCC Name “32D clone 3”). Theparental 32D cl3 cells were transfected with the cDNA for the human GCSFreceptor. The sequence for human GCSF receptor is M59818.GB_PR1(GenBank). The vector used to express this cDNA is called pLJ, seeKorman, Alan J., Frantz, J. Daniel, Strominger, Jack L., and Mulligan,Richard C., PNAS, 84:2150-2154 (1987). These cells were grown in RPMI1640, supplemented with 10% Fetal Bovine Serum, 10 ng/mL murine IL-3(mIL-3). A commercial source of mIL-3 is available from Biosource.

To transfect the 32D HuG-CSFR ck3 cells with the reporter nucleic acidconstruct, 30 μg of reporter nucleic acid construct was electroporatedinto 1×10⁷ 32D HuG-CSFR ck3 cells in a 4 mm cuvette at capacitance 500μF, 300V using an Electro Cell Manipulator EDM 600 (BTX). Cells wereincubated in nonselective medium overnight, then transferred intoselective medium (RPMI 1640, 10% Fetal Bovine Serum, 10 ng/mL mIL-3, and900 μg/mL Hygromycin B. Cells were plated at a density of approximately4.1 E5/well/mL in each well of a 24 well plate and left undisturbed for2 weeks. Actively growing colonies were pooled and divided into thewells of a six-well dish and then passaged in selective medium for twoweeks.

Single cell clones were established by limiting dilution in selectivemedium and clonal populations were individually tested for G-CSFresponsiveness. Specifically, clonal populations were incubated for fourhours with 0.8 ng/mL and 5.0 ng/mL GCSF. Best responders were identifiedbased on luminescence output-fold stimulation over background (assaymedium alone). One such population of cells, which showed a three-foldstimulation over background, was chosen and designated as HuG-CSFR-luc(clone 40) cells. This is a cloned, stably transfected cell line.

Example 5

Making UT7/9X Cell Line

To generate the reporter gene construct, a DNA fragment containing the9X response element repeat sequence was removed from pGTPLAP bydigestion with KpnI and SacII. The DNA fragment had the sequence (boththe strand shown and the complementary strand will have 3′ overhangsresulting from the digestion):

CGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCG CCGTACCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGCCGTACCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGTCATTTCCAGGAAATCA CCGC.

A linker comprising SacII, NheI, SmaI, XhoI, and BglII sites wassynthesized. A three-way ligation reaction was performed, comprised ofthe linker, the fragment containing the 9X response element repeatsequence, and pGL3 hygro that was cut with KpnI and BglII. This reactionserved to ligate the linker and the fragment containing the 9X responseelement repeat sequence into the pGL3 hygro. pGL3 hygro, which containsthe SV40 promoter, is described in Example 4 above. The resultingreporter nucleic acid construct was named pGL3 hygro 9X only #1.

To generate the pool population of UT7/9X cells, 1×10⁷ UT7/EPO cells(Komatsu, N. et al. (1993) Blood 82: 456-464) grown in IMDM supplementedwith 10% Fetal Bovine Serum (heat inactivated) and 1 U/mL of rHuEPO weretransfected by electroporation with 30 pg of pGL3 hygro 9X only #1 in a4 mm cuvette at capacitance 500 μF, 300V using an Electro CellManipulator ECM 600 (BTX). Cells were incubated in nonselective mediumovernight, then passaged into selection medium (IMDM, 10% Fetal BovineSerum, 1 U/mL rHuEPO, and 500 μg/mL Hygromycin B). Cells were plated andleft undisturbed for 2 weeks. Actively growing colonies were then pooledand passaged into plates. Single cell clones were established bylimiting dilutions. Clonal populations were individually tested for EPOresponsiveness. Specifically, clonal populations were incubated withthree different concentrations of rHuEPO (0.1, 1, and 3 U/mL). In theinitial evaluation, best responders were selected based on a ≧4 foldluminescence output fold stimulation over background (assay mediumalone). For the final selection of the clonal cell line, a full doseresponse curve was performed using Aranesp™ (range 200-0.01 ng/mL).

One such population of cells was chosen and designated as UT7/9X#6cells. UT7/9X#6 was selected based on demonstrating the highest foldstimulation over the linear range of sensitivity of Aranesp™ (5 fold)and the lowest background. This is a cloned, stably transfected cellline.

Example 6 Determining In Vitro Potency of Recombinant HumanErythropoietin (rHuEPO)

Wash UT7/9X cells with PBS twice. Resuspend cells in Assay Medium (247.5mL RPMI 1640 1X liquid with GlutaMAX™ and HEPES buffer, with phenol red,247.5 mL RPMI 1640 1X liquid without phenol red, 5 mL Fetal BovineSerum, filter through a 0.22 μM filter unit) to a concentration ofapproximately 4.0E+05 cells/mL and transfer to a horizontally positionedflask in a humidified incubator. Incubate at 37±2° C. and 5±1% for 17-24hours. Following this incubation, determine cell concentration andpercent cell viability. Viability should be 75%. In a 50 mL conicaltube, prepare a cell suspension from this cell preparation in AssayMedium to a final concentration of 2E+05 cells/mL. Mix well.

Dilute recombinant human Epo (rHuEPO) standard to approximately 40 ng/mL(4.8 U/mL) in Assay Medium (final concentration in wells will be 20ng/mL, 2.4 U/mL). Make 9 serial dilutions (1:2 is suggested) in AssayMedium to create a 10 point dose response curve. It is suggested toperform replicates of three for each dilution tested.

Add 25 μL of UT7/9XGAS cells that are prepared as described in the firstparagraph of Example 6 above to wells containing either 25 μL ofstandard or test sample. Wells to determine background contain 25 μL ofUT7/9XGAS cells with 25 μL of Assay Media. Cells are mixed frequently inreagent reservoir to ensure uniformity when adding to assay plates.Final cell concentration is approximately 5,000 cells/well.

Media Only wells are also created that contained only 50 μL of AssayMedia, containing neither rHuEPO nor cells. Plates are shaken and thenincubated for 4±0.5 hours in the humidified incubator at 37±2° C. and5±1% CO₂. Plates are removed and are allowed to come to room temperaturewithout the lid for a minimum of ten minutes, not to exceed twentyminutes.

Steady-Glo™ Luciferase Assay System (Promega #E2520) is used inaccordance with instructions. Add 50 μL of Steady-Glo™ to each well ofassay plates, including Media Only wells. Plates are covered to minimizelight exposure. Plates are shaken on plate shaker for a minimum of 5minutes. Plates are then incubated at room temperature for 10 minutes to2 hours. Alternatively, the plates may be left on the shaker for thisincubation period.

Plates are read in a Luminometer TopCount NXT Microplate Scintillationand Luminescence Counter (Packard/Perkin Elmer). Allow the plates toadapt in the dark for at least 1 minute before reading. Readluminescence for a minimum of 1 second per well. Import luminescencevalues into Excel or similar software package. Plot relativeluminescence units versus log dose concentration. Calculate effectiveconcentration 50 (EC₅₀) for standard and test samples.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein.

1. An isolated nucleic acid comprising a response element regioncomprising: (i) the sequence GTCATTTCCAGGAAATCACC or (ii) a sequencecomplementary to the sequence in (i).
 2. An isolated nucleic acidcomprising a response element region comprising: (a) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAA TCACCGTCATTTCCAGGAAATCACC or (ii) asequence complementary to the sequence in (i); (b) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC or (ii) a sequence complementaryto the sequence in (i); (c) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATC ACC or (ii) asequence complementary to the sequence in (i); or (d) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACC or(ii) a sequence complementary to the sequence in (i); wherein Y, X, andZ are each independently selected from a nucleic acid sequence of 0 to23 nucleotides.
 3. The isolated nucleic acid of claim 2, comprising thesequence: GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTT CCAGGAAATCACC

wherein Y, X, and Z are each a nucleic acid sequence of 0 nucleotides.4. The isolated nucleic acid of claim 2, comprising the sequence:GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTT CCAGGAAATCACC

wherein Y, X, and Z are each a nucleic acid sequence of 8 nucleotides.5. The isolated nucleic acid of claim 4, wherein Y, X, and Z are eachthe nucleic acid sequence GCCGTACC.
 6. The isolated nucleic acid ofclaim 2, comprising the sequence:GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTT CCAGGAAATCACC

wherein Y is a nucleic acid sequence of 8 nucleotides, X is a nucleicacid sequence of 10 nucleotides, and Z is a nucleic acid sequence of 16nucleotides.
 7. The isolated nucleic acid of claim 6, wherein Y is thenucleic acid sequence GCCGTACC, X is the nucleic acid sequenceTACCGGTCTG, and Z is the nucleic acid sequence ACCGGCCTAGTGCGTC.
 8. Theisolated nucleic acid of claim 2, comprising the sequence:GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC

wherein Y and X are each a nucleic acid sequence of 0 nucleotides. 9.The isolated nucleic acid of claim 2, comprising the sequence:GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC

wherein Y and X are each a nucleic acid sequence of 8 nucleotides. 10.The isolated nucleic acid of claim 9, wherein Y and X are each thenucleic acid sequence GCCGTACC.
 11. The isolated nucleic acid of claim2, comprising the sequence: GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC

wherein Y is a nucleic acid sequence of 8 nucleotides and X is a nucleicacid sequence of 10 nucleotides.
 12. The isolated nucleic acid of claim11, wherein Y is the nucleic acid sequence GCCGTACC and X is the nucleicacid sequence TACCGGTCTG.
 13. A vector comprising a promoter and nucleicacid comprising a response element region comprising: (i) the sequenceGTCATTTCCAGGAAATCACC or (ii) a sequence complementary to the sequence in(i).
 14. A vector comprising a promoter and nucleic acid comprising aresponse element region comprising: (a) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAA TCACCGTCATTTCCAGGAAATCACC or (ii) asequence complementary to the sequence in (i); (b) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC or (ii) a sequence complementaryto the sequence in (i); (c) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATC ACC or (ii) asequence complementary to the sequence in (i); or (d) (i) the sequenceGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACC or(ii) a sequence complementary to the sequence in (i); wherein Y, X, andZ are each independently selected from a nucleic acid sequence of 0 to23 nucleotides.
 15. The vector of claim 14, further comprising areporter nucleic acid, wherein the response element region is operablylinked to the promoter and the promoter is operably linked to thereporter nucleic acid.
 16. The vector of claim 15, wherein the promoteris a TK promoter and the reporter nucleic acid is a nucleic acid thatencodes luciferase.
 17. The vector of claim 15, wherein the promoter isa SV40 promoter and the reporter nucleic acid is a nucleic acid thatencodes luciferase.
 18. A host cell comprising a vector of any one ofclaims 13 to
 17. 19. The host cell of claim 18, wherein the host cell isa signaling molecule-responsive host cell.
 20. The host cell of claim18, wherein host cell is responsive to at least one signaling moleculeselected from G-CSF, EPO, and IL-3.
 21. The host cell of claim 20,wherein host cell is responsive to at least one signaling moleculeselected from recombinant methionyl human granulocyte colony-stimultingfactor, epoetin alfa, and darbepoetin alfa.
 22. A method for determiningthe activity of a test composition comprising a signaling molecule,comprising a) contacting the test composition with a signalingmolecule-responsive host cell comprising the vector of claim 15 underconditions in which the reporter nucleic acid expresses a reporterprotein in response to the signaling molecule; and b) detecting thereporter protein to determine the activity of the test composition. 23.The method of claim 22, further comprising comparing the level ofdetected reporter protein expression in (b) with the level of reporterprotein expressed by a signaling molecule-responsive host cellcomprising the vector of claim 15 in the absence of the signalingmolecule.
 24. The method of claim 22, further comprising comparing thelevel of detected reporter protein expression in (b) with the level ofreporter protein expressed by a signaling molecule-responsive host cellcomprising the vector of claim 15 in the presence of a standardcomposition comprising the signaling molecule.
 25. The method of claim24, further comprising calculating the relative potency of the testcomposition, wherein the relative potency is calculated by dividing thesignaling molecule concentration of the standard composition that givesa level of reporter protein expression of U by the signaling moleculeconcentration of the test composition that gives a level of reporterprotein expression of U.
 26. The method of any of claims 22 to 25,wherein the promoter is a TK promoter and the reporter nucleic acidencodes luciferase.
 27. The method of any of claims 22 to 25, whereinthe promoter is a SV40 promoter and the reporter nucleic acid encodesluciferase.
 28. The method of any of claims 22 to 25, wherein host cellis responsive to at least one signaling molecule selected fromG-CSF-like molecule, erythropoietic product, and IL-3.
 29. The method ofclaim 28, wherein host cell is responsive to at least one signalingmolecule selected from recombinant methionyl human granulocytecolony-stimulting factor, epoetin alfa, and darbepoetin alfa.
 30. Amethod for determining whether a test compound has activity of a givensignaling molecule, comprising a) contacting the test compound with asignaling molecule-responsive host cell comprising the vector of claim15 under conditions in which the reporter nucleic acid expresses areporter protein in response to compounds that have the activity of thegiven signaling molecule; b) detecting the reporter protein; c)comparing the level of detected reporter protein expression in (b) withthe level of detected reporter protein expressed by a signalingmolecule-reponsive host cell comprising the vector of claim 15 in theabsence of the test compound to determine whether the test compound hasthe activity of the given signaling molecule.
 31. The method of claim30, wherein the promoter is a TK promoter and the reporter nucleic acidencodes luciferase.
 32. The method of claim 30, wherein the promoter isa SV40 promoter and the reporter nucleic acid encodes luciferase. 33.The method of claim 30, wherein the given signaling molecule is selectedfrom G-CSF-like molecule, erythropoietic product, and IL-3.
 34. Themethod of claim 33, wherein the given signaling molecule is selectedfrom recombinant methionyl human granulocyte colony-stimulting factor,epoetin alfa, and darbepoetin alfa.
 35. A method for determining whethera test compound has activity of a given signaling molecule, comprisinga) contacting the test compound with a signaling molecule-responsivehost cell comprising the vector of claim 15 under conditions in whichthe reporter nucleic acid expresses a reporter protein in response tocompounds that have the activity of the given signaling molecule; b)detecting the reporter protein; c) comparing the level of detectedreporter protein expression in (b) with the level of detected reporterprotein expressed by a signaling molecule-responsive host cellcomprising the vector of claim 15 in the presence of the given signalingmolecule, but in the absence of the test compound, to determine whetherthe test compound has the activity of the given signaling molecule. 36.The method of claim 35, wherein the promoter is a TK promoter and thereporter nucleic acid encodes luciferase.
 37. The method of claim 35,wherein the promoter is a SV40 promoter and the reporter nucleic acidencodes luciferase.
 38. The method of claim 35, wherein the givensignaling molecule is selected from G-CSF-like molecule, erythropoieticproduct, and IL-3.
 39. The method of claim 38, wherein the givensignaling molecule is selected from recombinant methionyl humangranulocyte colony-stimulting factor, epoetin alfa, and darbepoetinalfa.
 40. A method for determining whether a test compound impacts theactivity of a signaling molecule, comprising a) contacting the testcompound with a signaling molecule-responsive host cell comprising thevector of claim 15 in the presence of the signaling molecule underconditions in which the reporter nucleic acid expresses a reporterprotein in response to the signaling molecule; b) detecting the reporterprotein; c) comparing the level of detected reporter protein expressionin (b) with the level of detected reporter protein expressed by asignaling molecule-reponsive host cell comprising the vector of claim 15in the presence of the signaling molecule, but in the absence of thetest compound, to determine whether the test compound impacts theactivity of the signaling molecule.
 41. The method of claim 40, whereinthe promoter is a TK promoter and the reporter nucleic acid encodesluciferase.
 42. The method of claim 40, wherein the promoter is a SV40promoter and the reporter nucleic acid encodes luciferase.
 43. Themethod of claim 40, wherein host cell is responsive to at least onesignaling molecule selected from G-CSF, EPO, and IL-3.
 44. The method ofclaim 43, wherein host cell is responsive to at least one signalingmolecule selected from G-CSF, epoetin alfa, and darbepoetin alfa.
 45. Amethod of producing a polypeptide from an ex vivo mammalian system,comprising producing the polypeptide, testing the polypeptide with thehost cell of claim 18, and determining the amount of protein producedand/or activity of the protein produced by the ex vivo system.
 46. Aresponse element region comprising more than one response elementsequences comprising the sequence GTCATTTCCAGGAAATCACC wherein thecenter region of at least two response element sequences are spatiallyoriented to be in the same location (on the y and z axis) plus or minus36 degrees, relative to the center axis of the double-helical DNA(x-axis), wherein the center region is the tenth and eleventhnucleotides AG of the sequence GTCATTTCCAGGAAATCACC.
 47. A responseelement region comprising more than one response element sequence coreregions comprising the sequence TTCCAGGAA wherein the center region ofat least two response element sequence core regions are spatiallyoriented to be in the same location (on the y and z axis) plus or minus36 degrees, relative to the center axis of the double-helical DNA(x-axis), wherein the center region is the fifth and sixth nucleotidesAG of the sequence TTCCAGGAA.
 48. A response element region comprisingat least two series of more than one response element sequencescomprising the sequence GTCATTTCCAGGAAATCACC; wherein each series ofmore than response element sequences are linked together by a sequenceof approximately eight nucleotides, wherein, within a first series ofthe response element sequences, each center region of the responseelement sequences are spatially oriented to be in approximately the samelocation (on the y and z axis) plus or minus 36 degrees, relative to thecenter axis of the double-helical DNA (x-axis), wherein the centerregion is the tenth and eleventh nucleotides AG of the sequenceGTCATTTCCAGGAAATCACC; wherein, within a second series of the responseelement sequences, each center region of the response element sequencesare spatially oriented to be in approximately the same location (on they and z axis) plus or minus 36 degrees, relative to the center axis ofthe double-helical DNA (x-axis), wherein the center region is the tenthand eleventh nucleotides AG of the sequence GTCATTTCCAGGAAATCACC; andwherein the center region of the response element sequences of thesecond series of the response element sequences are spatially orientedto be approximately 72 to 86 degrees from the center region of the firstseries of the response element sequences as determined from the y and zaxis relative to the center axis of the double-helical DNA as the xaxis.
 49. A response element region comprising at least two series ofmore than one response element sequences comprising the sequenceGTCATTTCCAGGAAATCACC; wherein each series of more than one responseelement sequences are linked together by a sequence of approximatelyeight nucleotides, wherein, within a first series of the responseelement sequences, each center region of the response element sequencesare spatially oriented to be in approximately the same location (on they and z axis) plus or minus 36 degrees, relative to the center axis ofthe double-helical DNA (x-axis), wherein the center region is the tenthand eleventh nucleotides AG of the sequence GTCATTTCCAGGAAATCACC;wherein, within a second series of the response element sequences, eachcenter region of the response element sequences are spatially orientedto be in approximately the same location (on the y and z axis) plus orminus 36 degrees, relative to the center axis of the double-helical DNA(x-axis), wherein the center region is the tenth and eleventhnucleotides AG of the sequence GTCATTTCCAGGAAATCACC; and wherein thecenter region of the response element sequences of the second series ofthe response element sequences are spatially oriented to beapproximately 144 to 180 degrees from the center region of the firstseries of the response element sequences as determined from the y and zaxis relative to the center axis of the double-helical DNA as the xaxis.
 50. The isolated nucleic acid of claim 2, wherein Y, X, and/or Zare independently selected from a sequence that is capable of binding toat least one transcription factor selected from _NFAT, AP-1, CRE, NFκB,and a member of the STAT protein family.